Functional Characterization of the Paeonia ostii P5CS Gene under Drought Stress

With persistent elevation in global temperature, water scarcity becomes a major threat to plant growth and development, yield security, agricultural sustainability, and food production. Proline, as a key osmolyte and antioxidant, plays a critical role in regulating drought tolerance in plants, especially its key biosynthetic enzyme, delta-1-pyrroline-5-carboxylate synthase (P5CS), which always positively responds to drought stress. As an important woody oil crop, the expansion of Paeonia ostii cultivation needs to address the issue of plant drought tolerance. Here, we isolated a PoP5CS gene from P. ostii, with an open reading frame of 1842 bp encoding 613 amino acids. PoP5CS expression progressively increased in response to increasing drought stress, and it was localized in the cytoplasm. Silencing of PoP5CS in P. ostii reduced drought tolerance, accompanied by decreased proline content, elevated reactive oxygen species (ROS) accumulation, and increased relative electrical conductivity (REC) and malondialdehyde (MDA) levels. Conversely, overexpression of PoP5CS in Nicotiana tabacum plants enhanced drought resistance, manifested by increased proline levels, reduced ROS accumulation, and lower REC and MDA contents. This study isolates PoP5CS from P. ostii and validates its role in regulating drought tolerance, providing valuable genetic resources and theoretical insights for the development of drought-resistant P. ostii cultivars.


Introduction
In the face of a warming planet and increasingly unpredictable weather patterns, drought has emerged as a pressing global threat to agricultural productivity and ecosystem stability.As a primary environmental stressor, drought significantly impacts plant growth and development, often leading to reduced yield and even plant death [1].To survive and thrive under these adverse conditions, plants have evolved intricate mechanisms of osmotic adjustment, which play a pivotal role in mitigating the deleterious effects of drought stress.Osmotic adjustment is a physiological response that involves the accumulation of compatible solutes, such as proline, glycine betaine, and soluble sugars, within plant cells to maintain turgor pressure and cellular homeostasis [2][3][4][5][6].Recent studies have underscored the complexity and versatility of these mechanisms, revealing that different plant species and even cultivars within the same species exhibit distinct patterns of coping abilities and osmotic adjustment in response to drought [7,8].
Among the various adaptive mechanisms employed by plants to mitigate the detrimental effects of drought, the accumulation of proline stands out as a pivotal response.Proline, a cyclic subamino acid, has garnered considerable attention in recent years for its multifaceted roles in conferring drought tolerance to plants.Its accumulation under drought stress is not merely a passive consequence of cellular dehydration but an active Plants 2024, 13, 2145 3 of 14 key osmotic regulation-associated proline biosynthesis gene, P5CS.We found a total of three differential P5CS genes (Unigene0006017, Unigene0000731, and Unigene0008549) (Table S1).Among them, Unigene0008549 had the highest FPKM value and the highest differential fold of 4.04, suggesting its positive response to drought stress.Then, the full length of Unigene0008549 was obtained by PCR, and its coding sequence was 1842 bp in length, encoding 613 amino acids.To resolve the evolutionary relationship between Unigene0008549 and other homologs, we downloaded eight P5CS proteins from the NCBI database and applied them to an evolutionary tree analysis with Unigene0008549.As shown in Figure 1A, Unigene0008549 clustered with A. thaliana AtP5CS1 and AtP5CS2 and shared 57% homology with them, indicating that Unigene0008549 belonged to the P5CS family, therefore named as PoP5CS.Multiple sequence comparison showed that there existed a high sequence similarity between PoP5CS and P5CS family members (Figure 1B).Subsequently, the expression profiles of PoP5CS in P. ostii leaves sampled on Days 0, 4, 8, and 12 after drought treatment were scrupulously examined by quantitative real-time PCR (RT-qPCR) analysis.As shown in Figure 2, the expression level of PoP5CS showed a continuous increase, which was 1.82 times higher than that of Day 0 on Day 4, 20.61 times higher than that of Day 0 on Day 8, and reached an astonishing multiplicity of 260.37 on Day 12.The above results were consistent with its upregulated expression pattern in the transcriptome database, which was further evidence of the importance of PoP5CS for P. ostii to respond to drought stress.The above results revealed that PoP5CS positively responded to drought stress.

PoP5CS Positively Responds to Drought Stress
Based on a drought-related transcriptome database of P. ostii [28], we worked to find drought-responsive genes that regulate drought tolerance in P. ostii.We focused on the key osmotic regulation-associated proline biosynthesis gene, P5CS.We found a total of three differential P5CS genes (Unigene0006017, Unigene0000731, and Unigene0008549) (Table S1).Among them, Unigene0008549 had the highest FPKM value and the highest differential fold of 4.04, suggesting its positive response to drought stress.Then, the full length of Unigene0008549 was obtained by PCR, and its coding sequence was 1842 bp in length, encoding 613 amino acids.To resolve the evolutionary relationship between Unigene0008549 and other homologs, we downloaded eight P5CS proteins from the NCBI database and applied them to an evolutionary tree analysis with Unigene0008549.As shown in Figure 1A, Unigene0008549 clustered with A. thaliana AtP5CS1 and AtP5CS2 and shared 57% homology with them, indicating that Unigene0008549 belonged to the P5CS family, therefore named as PoP5CS.Multiple sequence comparison showed that there existed a high sequence similarity between PoP5CS and P5CS family members (Figure 1B).Subsequently, the expression profiles of PoP5CS in P. ostii leaves sampled on Days 0, 4, 8, and 12 after drought treatment were scrupulously examined by quantitative real-time PCR (RT-qPCR) analysis.As shown in Figure 2, the expression level of PoP5CS showed a continuous increase, which was 1.82 times higher than that of Day 0 on Day 4, 20.61 times higher than that of Day 0 on Day 8, and reached an astonishing multiplicity of 260.37 on Day 12.The above results were consistent with its upregulated expression pattern in the transcriptome database, which was further evidence of the importance of PoP5CS for P. ostii to respond to drought stress.The above results revealed that PoP5CS positively responded to drought stress.

PoP5CS is Located in the Cytoplasm
In order to further reveal the expression characteristics of PoP5CS protein, we utilized Nicotiana benthamiana leaves for subcellular localization.We transiently transformed fusion vectors containing P5CS protein and enhanced green fluorescent protein (eGFP) into N. benthamiana leaves.The fusion vector expressed in the A. thaliana RPW8 2 gene (accession number: NM_114906.2) and mCherry fusion protein was used to indicate cytoplasmic localization signals.Following infiltration, fluorescent signals at 488 nm and 561 nm were elicited in N. benthamiana leaves to clarify the protein localization of PoP5CS.The emission of the eGFP signal, originating from the 35 S promoter-driven fusion construct of PoP5CS-eGFP, was observed within the cytoplasmic compartment of N. benthamiana leaves, where it co-localized with a cytoplasmic marker protein (Figure 3).This finding provided evidence that the subcellular localization of the PoP5CS protein occurs specifically within the cytoplasm of N. benthamiana leaves.

PoP5CS is Located in the Cytoplasm
In order to further reveal the expression characteristics of PoP5CS protein, we utilized Nicotiana benthamiana leaves for subcellular localization.We transiently transformed fusion vectors containing P5CS protein and enhanced green fluorescent protein (eGFP) into N. benthamiana leaves.The fusion vector expressed in the A. thaliana RPW8 2 gene (accession number: NM_114906.2) and mCherry fusion protein was used to indicate cytoplasmic localization signals.Following infiltration, fluorescent signals at 488 nm and 561 nm were elicited in N. benthamiana leaves to clarify the protein localization of PoP5CS.The emission of the eGFP signal, originating from the 35 S promoter-driven fusion construct of PoP5CS-eGFP, was observed within the cytoplasmic compartment of N. benthamiana leaves, where it co-localized with a cytoplasmic marker protein (Figure 3).This finding provided evidence that the subcellular localization of the PoP5CS protein occurs specifically within the cytoplasm of N. benthamiana leaves.

PoP5CS is Located in the Cytoplasm
In order to further reveal the expression characteristics of PoP5CS protein, we utilized Nicotiana benthamiana leaves for subcellular localization.We transiently transformed fusion vectors containing P5CS protein and enhanced green fluorescent protein (eGFP) into N. benthamiana leaves.The fusion vector expressed in the A. thaliana RPW8 2 gene (accession number: NM_114906.2) and mCherry fusion protein was used to indicate cytoplasmic localization signals.Following infiltration, fluorescent signals at 488 nm and 561 nm were elicited in N. benthamiana leaves to clarify the protein localization of PoP5CS.The emission of the eGFP signal, originating from the 35 S promoter-driven fusion construct of PoP5CS-eGFP, was observed within the cytoplasmic compartment of N. benthamiana leaves, where it co-localized with a cytoplasmic marker protein (Figure 3).This finding provided evidence that the subcellular localization of the PoP5CS protein occurs specifically within the cytoplasm of N. benthamiana leaves.

PoP5CS-Silenced P. ostii Show More Sensitivity to Drought
To investigate the function of PoP5CS in regulating drought tolerance, we obtained control plants (P.ostii transformed with pTRV), as well as PoP5CS-silenced P. ostii line 1 and line 2 based on the virus-induced gene silencing (VIGS) technique, and cultured them to the leaf expanding stage.Before drought treatment, there was no significant difference in plant Plants 2024, 13, 2145 5 of 14 phenotypes among these plants (Figure 4A).Prior to the drought treatment, we treated PoP5CS-silenced P. ostii line 2 with foliar proline spray for 3 days, and control plants (P.ostii transformed with pTRV) and PoP5CS-silenced P. ostii line 1 were sprayed with deionized water as controls.Next, the above P. ostii plants were subjected to drought treatment.DNA and total RNA were extracted from control plants (P.ostii transformed with pTRV) and PoP5CS-silenced P. ostii line 1 and line 2 on Day 15 after drought treatment for positive transgenic plant validation.As shown in Figure 4B, when DNA was amplified using P. ostii Ubiquitin-specific primers and pTRV1-specific primers, all experimental plants had a bright band, whereas when pTRV2-specific primers were used to amplify DNA, the pTRV band position differed in length from PoP5CS-silenced P. ostii line 1 and line 2. In addition, we quantified the expression level of PoP5CS in the above plants and found that the expression level of PoP5CS in PoP5CS-silenced P. ostii line 1 and line 2 decreased compared with pTRV by 35.45% and 30.23%.When they were exposed to drought treatment, the control plants (P.ostii transformed with pTRV) and proline-treated PoP5CS-silenced P. ostii line 2 maintained a normal growth condition and did not show a drought damage phenotype (Figure 4A).In contrast, PoP5CS-silenced P. ostii line 1 plants showed drooping stems and wilted leaves, which might be attributed to the suppressed expression of PoP5CS (Figure 4A).Then, several physiological experiments were applied to investigate the deeper response of PoP5CS to drought.As shown in Figure 5A,B, H 2 O 2 accumulation identified by diaminobenzidine (DAB) staining and O 2 •− accumulation identified by nitrotetrazolium blue chloride (NBT) staining were used to assess the extent of ROS accumulation in P. ostii leaves.Among them, PoP5CS-silenced P. ostii line 1 plants showed large areas of brown and blue coloration in the leaves, implying a severe degree of drought damage.In contrast, control plants (P.ostii transformed with pTRV) and PoP5CS-silenced P. ostii line 2 plants showed little or almost no coloration in their leaves.Moreover, leaf water content, relative electrical conductivity (REC), and malondialdehyde (MDA) content all showed low levels under normal conditions (Figure 5C-E).When they were exposed to drought, PoP5CSsilenced P. ostii line 1 exhibited a more substantial decrease in water content, while control plants (P.ostii transformed with pTRV) and PoP5CS-silenced P. ostii line 2 showed a smaller decrease (Figure 5C).REC and MDA contents increased to a high extent in PoP5CS-silenced P. ostii line 1 and to a lesser extent in control plants (P.ostii transformed with pTRV) and PoP5CS-silenced P. ostii line 2 (Figure 5D,E).The above results suggest that PoP5CS played a positive role in regulating drought tolerance in P. ostii.

Overexpression of PoP5CS Increases Drought Tolerance in Nicotiana tabacum
To further confirm the function of PoP5CS in plant drought resistance, we generated two dependent PoP5CS transgenic N. tabacum lines, named line 1 and line 2. T2 generations of N. tabacum were cultivated for drought treatment.Under normal conditions, there was no significant difference between wild-type (WT) and PoP5CS transgenic N. tabacum lines

Overexpression of PoP5CS Increases Drought Tolerance in Nicotiana tabacum
To further confirm the function of PoP5CS in plant drought resistance, we generated two dependent PoP5CS transgenic N. tabacum lines, named line 1 and line 2. T2 generations of N. tabacum were cultivated for drought treatment.Under normal conditions, there was no significant difference between wild-type (WT) and PoP5CS transgenic N. tabacum lines (Figure 6A).Then, WT and PoP5CS transgenic N. tabacum lines were subjected to drought treatment.DNA and total RNA were extracted from WT and PoP5CS transgenic N. tabacum lines on Day 10 after drought treatment for positive transgenic plant validation.As shown in Figure 6B, when DNA was amplified using Hygromycin (hygromycin label region of pCAMBIA1301 vector)-specific primers and PoP5CS-specific primers, only a bright band existed in the lanes of PoP5CS transgenic N. tabacum lines.In addition, we quantified the expression level of PoP5CS in the above plants and found that the expression level of PoP5CS in PoP5CS transgenic N. tabacum lines were 29.59-fold (line 1) and 47.89-fold greater (line 2) when compared with WT (Figure 6C).After passing through 10 days of drought treatment, PoP5CS transgenic N. tabacum lines grew well in comparison to the severe drought damage condition of WT.The corresponding physiological data also matched the phenotypic observations (Figure 7).Among them, ROS were over-accumulated in WT and the water content of WT was significantly reduced compared with PoP5CS transgenic N. tabacum lines, while REC and MDA contents were significantly increased in the WT (Figure 7A-E).The above results confirmed the results that PoP5CS could increase drought tolerance in plants.
drought treatment, PoP5CS transgenic N. tabacum lines grew well in comparison to the severe drought damage condition of WT.The corresponding physiological data also matched the phenotypic observations (Figure 7).Among them, ROS were over-accumulated in WT and the water content of WT was significantly reduced compared with PoP5CS transgenic N. tabacum lines, while REC and MDA contents were significantly increased in the WT (Figure 7A-E).The above results confirmed the results that PoP5CS could increase drought tolerance in plants.

PoP5CS Regulates Drought Tolerance by Promoting Proline Accumulation
To deeper investigate the effects of PoP5CS, plant proline contents were determined in the control plants (P.ostii transformed with pTRV), PoP5CS-silenced P. ostii, WT, and PoP5CS transgenic N. tabacum (Figures 5F and 7F).Under normal conditions, the proline content of the control plants (P.ostii transformed with pTRV) was significantly higher than the PoP5CS-silenced P. ostii lines with an average 1.95-fold difference.At the same time, the proline content of the WT was slightly lower than the PoP5CS transgenic N. tabacum.After drought treatment, proline contents were substantially increased in all of the above plants.In VIGS assay, the proline contents in the control plants (P.ostii transformed with

PoP5CS Regulates Drought Tolerance by Promoting Proline Accumulation
To deeper investigate the effects of PoP5CS, plant proline contents were determined in the control plants (P.ostii transformed with pTRV), PoP5CS-silenced P. ostii, WT, and PoP5CS transgenic N. tabacum (Figures 5F and 7F).Under normal conditions, the proline content of the control plants (P.ostii transformed with pTRV) was significantly higher than the PoP5CS-silenced P. ostii lines with an average 1.95-fold difference.At the same time, the proline content of the WT was slightly lower than the PoP5CS transgenic N. tabacum.After drought treatment, proline contents were substantially increased in all of the above plants.In VIGS assay, the proline contents in the control plants (P.ostii transformed with pTRV) and PoP5CS-silenced P. ostii line 2 were much higher than PoP5CS-silenced P. ostii line 1.In the overexpression assay, the proline contents in PoP5CS transgenic N. tabacum lines were much higher than the WT.Obviously, the proline content was positively correlated with the drought tolerance of plants.These findings suggest that PoP5CS increased the drought tolerance of plants by promoting proline accumulation.

PoP5CS is a Member of the P5CS Family
When plants are subjected to stress, a significant accumulation of proline occurs within their bodies, thereby enhancing their adaptive capacity to stress.As a crucial osmotic regulatory substance, proline plays a pivotal role in lowering the water potential of plant cells, strengthening their water absorption and retention capabilities, and thus safeguarding cellular osmotic balance and subcellular structural stability [29,30].P5CS genes, a key role in plant proline biosynthesis, have been reported in a variety of plant species, such as A. thaliana, Cajanus cajan, N. benthamiana, and Oryza sativa [31][32][33][34][35].In this study, we isolated the PoP5CS gene from P. ostii.The coding sequence of the PoP5CS gene was 1842 bp in length, encoding 613 amino acids.Liu and Wang [36] cloned the AmP5CS gene from Avicennia marina, and the coding sequence of AmP5CS was 2148 bp (encoded 715 amino acid protein).Yang et al. [37] isolated the SpP5CS gene from Stipa purpurea.SpP5CS contained a coding sequence of 2196 bp which encoded 731 amino acids.Then, a phylogenetic tree clustered PoP5CS into the branch containing A. thaliana AtP5CS1 and AtP5CS2.PoP5CS, Theobroma cacao TcP5CS, and Gossypium hirsutum GhP5CS were involved in multiple sequence comparison, and they had high sequence similarity.These indicated that PoP5CS is a typical P5CS family member.

PoP5CS Exhibits a Positive Response to Drought Stress Conditions
Exploring the expression patterns of genes under different environmental conditions can help to reveal their potential roles in cellular processes and involved metabolic pathways.P5CS genes play an important role in plant responses to a variety of abiotic stresses, such as drought, high temperature, and low temperature, largely determined by their expression response to different environments [32,38].In O. sativa, salt-tolerant variants of plants exhibited an elevated level of OsP5CS mRNA transcripts under conditions of high salinity, alongside an increase in proline content, compared to their expression under standard conditions [31].In reed canary grass, expression pattern analysis indicated an upregulation of B231P5CS transcripts in leaves subsequent to salt treatment (200 mM NaCl), with peak transcript abundance observed 12 h post-exposure, marking a 14.69-fold increase compared to untreated control plants [39].In Carica papaya, an investigation into the expression dynamics of CpP5CS2 under both low (7 • C)-and high (35 • C)-temperature stresses was conducted.The findings revealed that both stress conditions triggered an upregulation of CpP5CS2 expression during storage, with this enhancement preceding the accumulation of proline [38].In S. purpurea, the expression of the SpP5CS gene demonstrated a comprehensive responsiveness to a spectrum of stress conditions, encompassing cold, salt, and PEG stresses.Notably, a markedly significant upregulation of SpP5CS gene expression was observed specifically during soil drought conditions and upon subsequent rehydration treatment [37].Here, we analyzed the dynamic expression pattern of PoP5CS in P. ostii plants subjected to drought stress.After 4 days of drought treatment, the expression of PoP5CS reached 1.86 times that under untreated conditions, increased to 20.61-fold at Day 8, and remarkably surged to 260.37-fold at 12 days of drought treatment.Combining the above results, PoP5CS strongly responded to drought stress, and this response may bring about a large accumulation of proline.

PoP5CS Improves Plant Drought Resistance by Accumulating Proline Content
Plants exhibit resilience against drought stress through a diverse array of intricate mechanisms, with cellular osmoregulation emerging as a pivotal mode of physiological adaptation.Proline, a pivotal osmolyte and antioxidant, assumes a paramount role in safeguarding the maintenance of cellular osmotic equilibrium and modulating the delicate balance of ROS within plant cells, thereby contributing significantly to the plant's drought tolerance capabilities [9].With this in mind, we attempted to validate the drought tolerance function of the PoP5CS gene in plants as well as to analyze the regulatory mechanisms by which they modulate drought tolerance.Through the VIGS technology and overexpression system, we obtained PoP5CS-silenced P. ostii and PoP5CS-overexpressing N. tabacum and observed their phenotypic responses to drought stress as well as changes in key physiological indicators.After drought treatment, the drought tolerance of these transgenic plants showed a significantly positive correlation with the expression levels of PoP5CS.The indirect assessment of cellular membrane integrity can be quantified through the REC of cellular leakages [40].PoP5CS-silenced P. ostii had higher REC compared with the control, while exogenous spraying of proline reduced the REC of PoP5CS-silenced P. ostii.Similarly, PoP5CS-overexpressing N. tabacum demonstrated lower REC than the WT.MDA represents the degree of membrane lipid peroxidation, which indirectly reflects the degree of damage to the cell membrane system [41].MDA accumulation in PoP5CS-silenced P. ostii leaves was significantly higher than the control, while MDA in proline-treated PoP5CS-silenced P. ostii leaves was significantly lower than that in deionized water-treated PoP5CS-silenced P. ostii.Concordantly, PoP5CS-overexpressing N. tabacum accumulated considerably less MDA relative to the WT.In addition, we assessed the abundance of ROS accumulation in transgenic plants by DAB and NBT staining and found that ROS accumulation in PoP5CS-silenced P. ostii was significantly higher than that of the control, whereas exogenous spraying of proline reduced the level of ROS accumulation in PoP5CS-silenced P. ostii.Likewise, ROS barely accumulated in PoP5CS-overexpressing N. tabacum.In a previous study, Chen et al. [42] treated Phaseolus vulgaris PvP5CS transgenic A. thaliana with NaCl, and found that the REC of transgenic PvP5CS A. thaliana was significantly lower than control plants.In Saccharum officinarum, the P5CS gene is a potential drought tolerance target gene, and overexpression of SoP5CS resulted in lower MDA content compared with the WT [43].In our study, we also found that plant drought tolerance, as well as indicators of drought damage, showed a high degree of consistency with proline content, and that high proline accumulation ensured that plants maintained normal growth after drought stress, such as proline-treated PoP5CS-silenced P. ostii and PoP5CS-overexpressing N. tabacum.Similar results have been identified in multiple species, such as S. purpurea, Lilium regale, and N. benthamiana [33,37,44].Our study validated, for the first time, the positive function of PoP5CS, a key gene for proline biosynthesis, in mediating proline-mediated drought tolerance in P. ostii, providing insights into the improvement of drought tolerance in P. ostii.

Plant Materials and Drought Treatment
Potting soil mixed with loam, peat, and perlite (1:1:1) was used to cultivate one-yearold plants of P. ostii.After three-day continuous watering, P. ostii plants were subjected to natural drought in a greenhouse without water supplementation.P. ostii leaves were collected on Days 0, 4, 8, and 12 after drought treatment.For the VIGS assay, transgenic P. ostii plants at the leaf expanding stage were subjected to a 15-day-drought treatment.For the heterologous overexpression assay, the T2 generation of transgenic N. tabacum was subjected to a 10-day drought treatment.For the subcellular localization assay, N. benthamiana seeds were sown in the above potting soil and incubated under long sunlight (25 • C 16 h light/22 • C 8 h dark).

Gene Cloning, Multiple Sequence Alignment, and Phylogenetic Analysis
We first extracted total RNA from P. ostii leaves and then synthesized complementary DNA (cDNA) using EasyScript RT/RI Enzyme Mix (TransGen, Beijing, China).For PoP5CS gene cloning, specific primers (Table S2) were designed based on reference gene sequences derived from the P. ostii transcriptome database (SRA: SRP161474).The 2 × Phanta Max Master DNA (Vazyme, Nanjing, China) was applied to amplify the PoP5CS gene, and then, purified products were ligated into the 5 × TA/Blunt-Zero Cloning Mix (Vazyme, Nanjing, China) and amplified by Escherichia coli.The sequence was confirmed by Tsingke (Nanjing, China).For phylogenetic analysis, the amino acid sequence of PoP5CS was used to search its homologs in other species based on the E-value algorithm in the National Center for Biotechnology Information (NCBI) database.Also, the P5CS proteins from the model plant A. thaliana and several woody plants (Populus trichocarpa, Malus domestica, Pyrus × bretschneideri, and Vitis vinifera) for which a full genome sequence was available were also included in the phylogenetic tree analysis.MEGA7.0 was used for protein alignment by Clustal W, and a neighbor-joining phylogenetic tree was constructed by the bootstrap method with 1000 replications, p-distance, and pairwise deletion.Sequence comparison was performed by DNAMAN 6.0.

RT-qPCR Analysis
Total RNA from P. ostii leaves (including temporally drought-treated leaves and leaves with gene silencing) and N. tabacum leaves with gene overexpression was extracted.Then, cDNA for RT-qPCR analysis was synthesized by 5 × EasyScript All-in-One Su-perMix (TransGen, Beijing, China).NovoStart SYBR qPCR Super Mix (Novoprotein, Suzhou, China) was employed in conjunction with a BIO-RAD CFX Connect Optics Module (Bio-Rad, Hercules, CA, USA) to detect gene fluorescence signals, and the comprehensive experimental procedures can be found in Yu et al. [45].The 2 −△△Ct method was employed to derive the gene relative expression level.For normalization, P. ostii Ubiquitin (JN699053) and N. tabacum tubulin alpha chain (TUBA, NP_001312557.2) served as internal reference controls.The primers used here can be found in Table S2.

VIGS Assay
The VIGS approach utilizing the pTRV vectors was employed to transiently silence the PoP5CS gene in P. ostii.In the TRV-based VIGS system, pTRV1 and pTRV2 are the two key viral vector components, each of which has a different function and works together to achieve the gene silencing effect [46].pTRV2 is responsible for silencing specific genes, and pTRV1 is responsible for the replication and systematic spread of plasmids in plants.The nonconserved fragment of PoP5CS (from 20 bp to 338 bp) was fused into a pTRV2 vector with gene-specific primers (forward 5 ′ -AAGGTTACCGAATTCTCTAGAGTATGGATTAGCTTT GAATGTGGATC-3 ′ , reverse 5 ′ -CGTGAGCTCGGTACCGGATCCTGTTCAGCCAGCTTGA TGTGAC-3 ′ ); 100 ng pTRV2-PoP5CS, pTRV2 and pTRV1 vectors were transformed into A. tumefaciens, respectively.A. tumefaciens cultures were cultured overnight in a shaker at 28 • C to achieve an OD of 1.5.The resuspended A. tumefaciens bacterial solution mixtures (pTRV2-PoP5CS mixed with pTRV1, pTRV2 mixed with pTRV1) were kept in the dark for 1 h.P. ostii plants at the dormant stage with 2~3 buds were used as infestation materials.After pruning to create wounds, the roots were washed 2~3 times using deionized water.Then, the roots were immersed in the above bacterial solution, and a vacuum desiccator was employed to create negative pressure for 30 min, facilitating the penetration of the bacterial solution into the plants.Subsequent to being washed twice with deionized water, the plants were repotted into potting soil, with each experimental group comprising 15 individual plants.After a period of 30 days under cultivation, the plants had reached the leaf expansion stage.For the drought tolerance assays, the control plants (P.ostii transformed with pTRV) and PoP5CS-silenced P. ostii line 1 plants were treated with deionized water, and PoP5CS-silenced P. ostii line 2 was treated with proline for 3 days to test the function of proline on P. ostii drought resistance following approximately 15 days of drought treatment.The primers used here can be found in Table S2.

Heterologous Overexpression Assay
The PoP5CS coding sequence was amplified by gene-specific primers (forward 5 ′ -CAGGTCGACTCTAGAGGATCCATGGCTCTTTATGATACTTTGTTCAGC-3 ′ , reverse 5 ′ -CGATCGGGGAAATTCGAGCTCCGACAACAGTGGGAGATCCTTG-3 ′ ) and then fused into a pCAMBIA1301 vector.The pCAMBIA1301-PoP5CS vector was introduced for N. tabacum transformation using the leaf disc method via T-DNA insertion as previously described [47].Briefly, the pCAMBIA1301-PoP5CS vector was transformed into A. tumefaciens; the A. tumefaciens bacterial solution was resuspended in infiltration buffer (10 mM MES, 10 mM MgCl 2 , and 0.2 mM acetosyringone) and adjusted to a standard concentration (OD = 0.3).Then, we took leaves from N. tabacum sterile seedlings, infiltrated the leaves using the above mentioned bacterial solution, filtered out the solution, and co-cultivated it for 3 days in darkness.The leaves were then transferred to resistant shoot screening and differentiation medium for selection culture.The differentiated adventitious shoots were transferred to rooting screening medium for rooting culture to obtain the T0 generation.Positive PoP5CS transgenic N. tabacum was screened by antibiotic labeling, and two independent lines of PoP5CS transgenic N. tabacum (T2 generation) were used to verify their function in drought tolerance.Positive PoP5CS transgenic N. tabacum seedlings rooted in the medium were transplanted into potting soil to carry out conventional greenhousebased cultivation.After approximately 3 months, PoP5CS transgenic N. tabacum plants with similar phenotypes were subjected to a 10-day natural drought treatment as above to verify their drought resistance function.The primers used here can be found in Table S2.

Physiological Index Measurement
DNA was first extracted from PoP5CS transgenic P. ostii leaves and PoP5CS transgenic N. tabacum leaves.Then, PCR and RT-qPCR were applied for positive plant validation (Table S2).Both leaves from PoP5CS transgenic P. ostii and PoP5CS transgenic N. tabacum before and after drought treatment were used for physiological index measurement.The specific experimental procedures of water content determination, REC measurement, MDA content measurement, and DAB and NBT staining were followed as previously described [48].The proline content was measured using a reagent kit (Comin Biotechnology Co., Ltd., Suzhou, China).Briefly, 0.1 g of PoP5CS transgenic P. ostii leaves and PoP5CS transgenic N. tabacum leaves were extracted by 1 mL sulfosalicylic acid solution.After homogenization in an ice bath, the mixture was subjected to oscillation extraction at 90 • C for 10 min, followed by centrifugation at 10,000× g and 25 • C for 10 min, with the supernatant being collected.To 0.5 mL of the supernatant, 0.5 mL of glacial acetic acid and 0.5 mL of reaction mixture (containing ninhydrin, glacial acetic acid, and concentrated phosphoric acid solution) were added.After thorough mixing, the solution was kept in a boiling water bath for 30 min.After cooling, 1 mL of toluene was added to the test tube, followed by shaking for 30 s.The absorbance at a wavelength of 520 nm was measured to calculate the proline content.

Statistical Analysis
All data consisted of at least three biological replicates and error bars indicate standard error.The significant differences were analyzed by one-way ANOVA (p < 0.05) or by Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 2 .
Figure 2. RT-qPCR analysis of the expression levels of PoP5CS under drought stress conditions on Days 0, 4, 8, and 12. PoP5CS expression on Day 0 is normalized to 1. Data represent the mean ± SD of three biological replicates, and letters indicate significant differences by one-way ANOVA (p < 0.05).

Figure 2 .
Figure 2. RT-qPCR analysis of the expression levels of PoP5CS under drought stress conditions on Days 0, 4, 8, and 12. PoP5CS expression on Day 0 is normalized to 1. Data represent the mean ± SD of three biological replicates, and letters indicate significant differences by one-way ANOVA (p < 0.05).

Figure 2 .
Figure 2. RT-qPCR analysis of the expression levels of PoP5CS under drought stress conditions on Days 0, 4, 8, and 12. PoP5CS expression on Day 0 is normalized to 1. Data represent the mean ± SD of three biological replicates, and letters indicate significant differences by one-way ANOVA (p < 0.05).

Figure 3 .
Figure 3. Subcellular localization of PoP5CS in Nicotiana benthamiana leaves.(a,e) The green fluorescence signals of the 35S:: eGFP expression vector and 35S:: PoP5CS-eGFP expression vector at 488 nm; (b,f) the red fluorescence signals of the RPW8 2-mCherry expression vector at 561 nm; (c,g) bright field images; (d) an overlaid image of (a-c); (h) an overlaid image of (e-g).

Figure 6 .
Figure 6.Phenotypic observation and positive plant identification of PoP5CS-overexpressing Nicotiana tabacum.(A) Phenotypic observation of PoP5CS-overexpressing N. tabacum when PoP5CS was overexpressed.Bar = 10 cm.(B) PCR validation of PoP5CS-overexpressing N. tabacum after 10 days of drought treatment.NtTUBA, specific primers in N. tabacum tubulin alpha chain coding sequence; Hyg, specific primers in the hygromycin label region of pCAMBIA1301 vector; PoP5CS, specific primers in PoP5CS coding sequence.(C) RT-qPCR validation of PoP5CS-overexpressing N. tabacum after 10 days of drought treatment.Data represent the mean ± SD of three replicates, and statistical significance was determined by Student's t-test (*** p < 0.001).

Figure 6 .
Figure 6.Phenotypic observation and positive plant identification of PoP5CS-overexpressing Nicotiana tabacum.(A) Phenotypic observation of PoP5CS-overexpressing N. tabacum when PoP5CS was overexpressed.Bar = 10 cm.(B) PCR validation of PoP5CS-overexpressing N. tabacum after 10 days of drought treatment.NtTUBA, specific primers in N. tabacum tubulin alpha chain coding sequence; Hyg, specific primers in the hygromycin label region of pCAMBIA1301 vector; PoP5CS, specific primers in PoP5CS coding sequence.(C) RT-qPCR validation of PoP5CS-overexpressing N. tabacum after 10 days of drought treatment.Data represent the mean ± SD of three replicates, and statistical significance was determined by Student's t-test (*** p < 0.001).Plants 2024, 13, x FOR PEER REVIEW 8 of 14

Figure 7 .
Figure 7. Physiological index measurement of PoP5CS-overexpressing Nicotiana tabacum.(A) H 2 O 2 accumulation condition of PoP5CS-overexpressing N. tabacum leaves after 10 days of drought treatment.Bar = 3 cm.(B) O 2 − accumulation condition of PoP5CS-overexpressing N. tabacum leaves after 10 days of drought treatment.Bar = 3 cm.(C) Leaf water content when PoP5CS was overexpressed.(D) REC when PoP5CS was overexpressed.REC, relative electrical conductivity.(E) MDA content when PoP5CS was overexpressed.MDA, malondialdehyde.(F) Proline content when PoP5CS was overexpressed.Data represent the mean ± SD of three replicates, and letters indicated significant differences by one-way ANOVA (p < 0.05).

Author Contributions:
Conceptualization, J.T.; methodology, J.T. and D.Z.; software, Y.L., H.A. and Z.C.; data curation, Y.L., H.A. and Z.C.; writing-original draft preparation, Y.L.; writing-review and editing, Y.L., J.T. and D.Z.; supervision, J.T. and D.Z.; project administration, J.T. and D.Z.; funding acquisition, J.T. and D.Z.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the Long-term scientific research base for conservation and innovative utilization of Peony germplasm resources in Jiangsu Province (LYKJ[2021]01), the Central finance forestry science and technology promotion demonstration fund project (Su[2023]TG05), the Agriculture Science and Technology Innovation Foundation of Jiangsu Province (CX[22]3186), the High-Level Talent Support Program of Yangzhou University, and National Forest and Grass Science and Technology Innovation and Development Research Project (2023132012).